Tissue slices are a common in vitro model to investigate physiological cell function. This old experimental approach is routinely used in neurophysiology, however, in heart research the concept failed in initial trials mainly because of the low survival time of slices. Nevertheless, a successful slice model from the heart would be of substantial interest for several reasons. (1)  From one heart a large number of slices can be obtained for experimental use whereas in the established model of papillary muscle or Purkinje fibers only very few preparations (1-3) are possible. (2) A transmural slice is a representative part of the heart and it is possible to study regional differences in action potential shapes within the ventricular wall. (3) In contrast to the in vitro model of enzymatically isolated myocytes, in slices the different cell types (myocytes, fibroblast etc.) remain in their anatomical context and the interaction between myocytes and non-myocytes can be studied. (4) Tissue slices could be an appropriate scaffold to study stem-cell formation and implementation.

A primary step is to validate the model and we have shown that from 300 µm thick tissue slices of the left ventricle of guinea-pig hearts (Figure 1) normal action potentials can be recorded with conventional intracellular glass electrodes over hours and pharmacological effects are similar to those obtained in the established papillary muscles preparation (Figure 2). Even non-invasive optical measurements with potential-sensitive dyes are possible. Electrical activity can also be recorded with a planar grid of extracellular electrodes (multielectrode array, MEA). Although the signals which can be obtained in this way are tiny in amplitude they allow to determine action potential duration and also spread of excitation within the slice. The mean propagation velocity was close to that of other cardiac preparations (Figure 3). Cardiac tissue slices can of course also be obtained from models of heart disease and from human specimen. Therefore this approach will be valuable for investigation of remodeling processes in cardiac diseases. In summary, the cardiac slice model is an important and promising new approach in cardiac research.

Bussek A, Wettwer E, Christ T, Lohmann H, Camelliti P & Ravens U. Tissue slices from adult mammalian hearts as a model for pharmacological drug testing. Cellular Physiology and Biochemistry (2009, in press)

de Boer T*, Camelliti P*, Ravens U & Kohl P. Myocardial tissue slices:
organotypic pseudo-2D models for cardiac research & development. Future Cardiology 5: 425-30 (2009) (*equal 1st author contribution)

Figure 1

Figure 2

Figure 3

'Spotlight' Articles Archive

July 2009: Atrial electrophysiological remodelling during atrial fibrillation

Electrical remodeling is associated with a shortening of effective refractory period (ERP) that predominantly results from abbreviation of action potential duration (APD) and with a loss of physiological rate adaptation of APD. Studies in patients with chronic AF (cAF) showed that decreased L-type Ca2+ current (ICa,L) and presumably reduced transient outward K+ current (Ito) are major contributors to shortening of APD. Additional work provided clear evidence for involvement of increased inward rectifier K+ currents IK1 and especially of a constitutively active IK,ACh. Recently the enhanced expression and function of the sodium-calcium exchanger (NCX) was also found during AF. The abbreviation in atrial refractoriness is not homogeneous which increases the likelihood of conduction block and wave break to occur. The wavelength, i.e. the product of ERP and conduction velocity, decreases thereby facilitating the development of functional reentry which maintains AF.

Better understanding the mechanisms responsible for electrical remodelling in atria would help to identify new putative targets for future antiarrhythmic therapy.

April 2009: Atrial myofibril remodelling in chronic atrial fibrillation

Recent investigations high-light the role of sarcomere protein modifications in the atrial contractile dysfunction associated to chronic atrial fibrillation (cAF). However, the functional impact of myofilament protein changes in human cAF is weakly documented because it is difficult to obtain consistent measurements of functionally relevant parameters on human cardiac preparations.

Studies on single myofibrils can significantly document changes in the mechanical performance of human cardiac sarcomeres because these preparations can be obtained in large amounts from very small cardiac samples. Single myofibrils are the smallest units of the contractile apparatus that retain the organized myofilament lattice and its entire ensemble of associated proteins. Mechanical measurements of myofibril force combined with rapid perfusion switching techniques have been developed recently to investigate fast kinetic events related to cross-bridge action and regulation in human cardiac myofibrils (Piroddi et al. Pflugers Arch. 454, 63-73, 2007).

To dissect the impact of cAF on myofilament function (eliminating changes induced by the arrhythmia in atrial myocyte membranes and extracellular components) we isolated myofibrils from atrial samples of 10 patients in sinus rhythm (SR) and 12 patients with cAF undergoing corrective cardiac surgery. Active tension changes following fast increase and decrease in [Ca2+] and the sarcomere length-passive tension relation were determined in the two groups of myofibrils. Compared to SR myofibrils, cAF myofibrils showed (i) a reduction in maximum tension and in the rates of tension activation and relaxation; (ii) an increase in myofilament Ca2+-sensitivity of tension; (iii) a reduction in myofibril passive tension. The slow β-myosin heavy chain (MHC-β) isoform and the more compliant titin isoform N2BA were up regulated in cAF as compared to SR atrial myocardium. Alterations in active and passive tension generation at the sarcomere level, explained in part by changes in MHC and titin isoforms, are part of the contractile dysfunction of cAF and may contribute to the self-perpetuation of the arrhythmia and the development of atrial dilatation (Belus et al, submitted).

The initial and steady-state sarcomere remodeling following the onset of AF is now tested in myofibrils isolated from chronically instrumented animal models of the arrhythmia (work in progress).

Single myofibril technique: Right, myofibrils (diameter 1-2 μm) are mounted between a cantilever force probe (down) and a glass needle (up) mounted on the lever arm of a length control motor. Force is measured photoelectronically by recording the force probe deflection.  Left, myofibrils are activated and relaxed by rapid solution switching between two continuous streams of solutions.

November 2008: HCN channels - What are they good for in the heart?

The heart beat is triggered by an ensemble of highly specialized cells in the sinoatrial node (SAN) that has the unique property to contract spontaneously and rhythmically in the absence of external stimuli. These cells –also called pacemaker cells- produce a specific kind of action potential that differs from action potentials of normal cardiomyocytes by the presence of a diastolic depolarization (DD). The DD is initiated at the end of the repolarization phase of the action potential and depolarizes the membrane to the threshold required for triggering the next action potential. There is good evidence that the “funny” current (If) that was discovered in the late seventies by DiFrancesco and his colleagues plays a pivotal role in the generation of the DD. If has several unique properties that predestinate this current to contribute to the DD. Most importantly, the current is activated upon hyperpolarization and is enhanced by cAMP in a direct, protein phosphorylation-independent fashion. The latter feature plays a crucial role in the up- and down-regulation of heart rate by sympathetic and vagal stimulation, respectively.

It took another 20 years after the first discovery of If to identify the proteins underlying this current. The proteins, now designated as HCN channels, were discovered independently by three groups in Germany and USA. HCN channels display the principal hallmark features of native If. Notably, these channels like native If are gated by both hyperpolarization and cyclic AMP. The term “HCN” which stands for “hyperpolarization-activated cyclic nucleotide-gated” refers to this unique activation profile. HCN channels comprise a small gene family consisting of four members (HCN1-4). While all four channels are expressed in heart tissue, HCN4 is the predominant channel in SAN cells and confers the major fraction of If. Pharmacological block of this channel by specific agents, such as ivabradine, has emerged as a novel therapeutic approach to lower heart rate in cardiovascular diseases such as stable angina pectoris. There is also increasing evidence that expression of HCN channels outside of the SAN region (so-called ectopic expression, e.g. in ventricular muscle) is part of the pathology of several heart diseases (e.g. heart failure). Ectopic HCN channel expression may contribute substantially to the generation of severe arrhythmia leading to sudden death. Thus, blockade of ectopic HCN channels may be also beneficial for patients.

August 2008: How is contraction of the heart triggered?

The major role of the heart is to develop pressure to pump blood throughout the body. The required force is generated by muscle proteins. It is important that the heart relaxes between beats so that it can refill with blood. Furthermore the force of contraction needs to be greater in exercise than at rest. These considerations require that contraction is regulated and this is done using changes of the concentration of calcium in the cell. On each heartbeat, therefore, there is an increase of calcium concentration inside the cell (the so called “calcium transient”). This calcium can be measured with calcium-sensitive indicators inserted into the cell (see movie below). We now know that most of this calcium is kept within a store inside the cell (the sarcoplasmic reticulum) and is released through a specialized channel known as the Ryanodine Receptor (RyR).

July 2008: How the heart beats

Beating about 100,000 times a day at a regular rhythm for some 2-3 billion times in an average lifetime makes the human heart one of the most efficient and performing biological machines. This extraordinary performance relies on the heart’s ability to beat spontaneously, without external stimuli. Indeed, while innervation by the autonomic nervous system is essential for modulation of rate, spontaneous activity is an intrinsic function of cells from the pacemaker region, the sino-atrial node (SAN). How do SAN cells beat spontaneously? In 1979 Hilary Brown, Susan Noble and Dario DiFrancesco described for the first time a “funny” (If) current from a mammalian SAN preparation with properties which, while unusual, were perfectly fit to explain spontaneous activity and adrenaline-induced rate acceleration.

If was an inward current activated on hyperpolarization to the diastolic range of voltages, properties expected for a mechanism able to generate and control the “diastolic depolarization” phase of the action potential, which in SAN cells is responsible for repetitive activity. Following this original report, 30 years of intense investigation have established the role of funny current in cardiac pacemaking and in the autonomic modulation of heart rate.

More recently, the concept of funny channel-based pacemaking has been applied to practical developments of clinical relevance. These include the “biological” pacemakers, the application to a genetic approach to rhythm disorders caused by dysfunctional funny channels, and most importantly the development of the drug ivabradine, which slows heart rate by specific inhibition of funny channels and is therefore prescribed against chronic stable angina. Whereas the funny current is far from being the only cellular mechanism necessary to pace the cell, its role makes it an ideal target to improve our understanding of pacemaker activity and use this knowledge in clinically-relevant applications.

May 2008: Computational physiology research: the European Virtual Human initiative

The approach of the normaCOR project, combining 'wet' and 'dry' research, has recently received a major boost by the EC Framework 7 focus on the Virtual Physiological Human (VPH).

The VPH is aimed at making optimal use of the wealth of data provided by modern laboratory techniques. Using a 'feedback loop', physiological data is used to create models that allow researchers to test hypotheses in silico, before model predictions are validated in new clinical or experimental research.  These virtual experiments enable researchers to assess and test their assumptions and, in addition, help to refine and partially replace animal experimentation.  Quantitative computer models can be used at all levels of complexity, from molecule to man. They already aid the research in the normaCOR projects, and will in future help clinicians in information gathering, sharing, interpretation, and thereby support decision making about individual patient care.

This approach to physiology research is gaining momentum.   In addition to the work of individual research groups around the world, the field of computational biology has benefited from coordination and funding at a European level under the EC’s Sixth and Seventh Framework programmes.  The STEP project, 2005/06 initiated the community building of researchers from various backgrounds, working towards the Virtual Physiological Human. The success of this project, and the resultant VPH Roadmap, laid basis to the creation of the Virtual Physiological Human Network of Excellence, coordinated by jointly by the Oxford normaCOR partner and the University College London. This Network will 'kick off' in June 2008. 

In total, 14 VPH projects were funded under the same Information and Communication Technologies call, including two cardiac modelling projects that are co-lead by Oxford.  In this way, the understanding of arrhythmogenic mechanisms, as well as the advanced computational techniques, gained by the normaCOR project will be carried forward under FP7 by preDiCT which aims to model and predict the arrhythmogenic properties of drug candidates, and euHeart which seeks to develop simulation tools to aid doctors making clinical decisions.

April 2008: Dario DiFrancesco (Milan team) wins Grand Prix, Fondation LeFoulon-Delalande

Dario DiFrancesco (now in Milan) has been awarded the Grand Prix of the Fondation LeFoulon-Delalde, awarded by the Institut de France, in Paris.  The prize for 2008 has been awarded to him for his discovery in Oxford of the i(f) current (published with Hilary Brown and Susan Noble in Nature in 1979), identifying its ion specificity, measuring its single channel conductance, computing its role in pacemaker rhythm (with Denis Noble), and characterising its biochemical control - leading to the creation of ivabradine (Servier), the first i(f) blocker, which has been approved for the treatment of angina.  This seems to be the first major international prize to be awarded for the discovery of a cardiac ion channel.